Nuclear magnetic resonance (NMR) is a popular tool for studying the physical, chemical and biological properties of matter at a molecular level, due to the flexibility and analytical capability of the technique. For example, NMR imaging techniques are routinely used by chemists to determine the structure of complicated molecules. Such techniques complement traditional x-ray crystallographic as a method for determining smaller protein structures of 25 kDa or less.
NMR takes advantage of the measurable behavior of the nuclei of certain atoms, when placed in a static magnetic field. Most nuclei possess a non zero spin and thus have a nuclear magnetic moment. In a semi-classical treatment, as the positively charged nucleus spins, the moving charge creates a magnetic moment.
When no external magnetic field is applied, the magnetic moments of nuclei are aligned randomly. However, if the nuclei are placed in an external homogeneous magnetic field (B0), the magnetic moments will either align with the external magnetic field or in opposition to the magnetic field. The alignment of the groups according to one of the two possible orientations follows Boltzmann's statistics and results in a population imbalance among the different energy levels and a net nuclear magnetization M. Accordingly, there will be slightly more nuclei at the lower energy level than at the higher energy level.
Because nuclei behave like magnets, the nuclei have a lower energy state when aligned with the applied magnetic field than when the nuclei are opposed to the magnetic field. A nucleus in the low energy state may transition to a high-energy state by the absorption of a photon that has an energy that is exactly equal to the energy difference between the two energy states. The energy of a photon is related to its frequency by Plank's constant. The frequency of the photon and the equivalent frequency of precession are referred to as the resonance or Larmor frequency.
Thus, it is possible to make magnetic dipoles “flip” from the low energy, more stable alignment to the high energy, less stable alignment by supplying the right amount of energy. The energy necessary to make this transition depends on the strength of the external magnetic field used and is usually in the range of energies found in radio waves. Therefore, the nuclei can absorb and reemit energy at characteristic radiofrequencies (RF). Furthermore, energy will be absorbed by the same nuclear species at slightly different frequencies depending on the molecular environment of the nucleus of a particular atom.
The precise resonant frequency of the nuclear species is dependent on the magnetic field at the nucleus, and will vary depending on the types of nuclei and the bonds in the molecule involving the nuclei. This characteristic variance in the resonance frequency depending on the chemical environment of the nucleus is called the chemical shift (δ) and can be used to deduce the patterns of atomic bonding in the molecule. In particular, the chemical shift is the frequency difference between the observed resonance and a resonance from a standard compound and is usually reported in parts per million (ppm) of the mean resonance frequency.
In the typical NMR experiment, a sample to be analyzed is placed in a homogeneous static external magnetic field (B0). By convention, B0 and the net magnetization vector (Mz) reside on the z-axis at equilibrium. Also by convention, a rotating frame of reference rotating around the z-axis at the Larmor frequency allows B0 and net nuclear magnetization M to appear static, i.e., the x′ and y′ axes rotate about the z-axis.
Accordingly, an applied radio frequency (RF) pulse has a stationary field vector in the xy plane within this reference frame with a direction governed by the phase of the radio frequency. The application of an RF pulse along the x-axis rotates the nuclear magnetization vector towards the y-axis at an angle that is proportional to the duration and intensity of the RF pulse. A pulse that is of sufficient duration and intensity to rotate the magnetization vector clockwise 90 degrees about the x-axis is termed a 90 degree (90°) or π/2 pulse. Similarly, a 180° pulse will rotate the magnetization vector 180 degrees and is called a π pulse.
Predictably, the populations of nuclei relax to equilibrium at an exponential rate after the termination of the applied RF pulse. Once the magnetization vector is placed onto the y-axis, it rotates in the xy plane at a resonant frequency ultimately decaying back to the z-axis emitting RF radiation over time. This is typically the point of data acquisition. A receiver coil resonant at the Larmor frequency, generally located along the x-axis, can detect this rotation, which is commonly referred to as the free induction decay (FID). Fourier transformation of the FID provides the NMR spectrum.
One time constant used to describe this return to equilibrium is called the longitudinal or spin lattice relaxation time (T1). The time (T1) will vary as a function of the magnetic field strength. A second time constant, known as the spin-spin relaxation time (T2), which is due to the exchange of energy between spins, is a description of the return to equilibrium of the transverse magnetization (Mxy) and is always equal to or less than T1.
A spin-echo pulse sequence is normally required to measure T2. The typical pulse-sequence consists of the application of a 90° pulse, which results in the rotation of the magnetization to the xy plane, followed by a 180° pulse that allows the magnetization to partially rephase producing a non-dephased signal called an echo.
In general, the practice of high-resolution NMR spectroscopy yields information about molecular structure and dynamics through the observation of interactions such as chemical shifts and scalar, dipole, quadrupole coupling interactions and the like. These features make NMR a powerful analytical tool, particularly for the study of dynamic processes such as the metabolism of plants and organisms, and the dynamics of geological processes, as well as for characterization of technologically important new materials.
One of the main disadvantages of NMR, however, is its limited sensitivity. The amount of sample necessary to get a readable signal is invariably fairly large, because in conventional NMR, the signal to noise ratio is proportional to the nuclear magnetic polarization of the sample. In addition, despite steady progress in the construction of stronger superconducting magnets, the nuclear magnetic polarization is only a small fraction of the attainable maximum (˜10−4 for protons in a 14 T magnet). Typically, 1016–1018 molecules are necessary for meaningful measurements. For a liquid or a solid, therefore, one needs under normal circumstances not less than a 1 mm3 sample.
Also, in conventional NMR, the detected signal results from a measurement of Faraday induction in the coil surrounding the sample, by monitoring the current induced in a tank circuit tuned to resonance. Compared to other detection methods, the sensitivity of this scheme is rather poor since, even with maximum polarization, the minimum number of spins needed to induce a measurable signal is invariably large. As a result, the spatial resolution currently achievable in Magnetic Resonance Imaging (MRI) or spectroscopy is limited to about 5 to 10 μm which represents a very important disadvantage when compared to microscopy techniques, e.g., near field scanning optical microscopy, scanning tunneling microscopy and transmission electron microscopy.
Accordingly, a need exists for a high resolution, high sensitivity NMR apparatus and method that can provide both spatial and spectroscopic data for a sample. The present invention satisfies those needs, as well as others, and generally overcomes the deficiencies found in the background art.